Analogs which utilize the catalytic mechanism of an enzyme (e.g. transition-state inhibitors) have been suggested as enzyme inhibitors, however it has only been recently that this idea has been explored. A major problem has been the difficulty in synthesizing the target peptide analog molecules. One candidate group are analogs having the aldehyde group. These peptide analogs are particularly attractive in that they can be prepared from naturally occurring amino acids. Highly specific and potent peptide transition-state analog enzyme inhibitors would be of interest as therapeutic agents. Solution methods for the synthesis of peptide aldehydes have been developed, however, their preparation remains a tedious, labor intensive, and time consuming process. The development of a methodology for the automated synthesis of these aldehyde derivatives will allow for the rapid synthesis of a large number of these analogs and, thus, would facilitate the exploration of enzyme inhibition structure-activity relationships. The availability of such analogs would facilitate the development of drugs that selectively inhibit specific serine or cysteine proteinases.
The serine proteinases may be suitable targets for inhibition by peptide transition-state analogs. The trypsin sub-family is composed of serine proteinases which hydrolyse peptide bonds that follow an arginine or lysine residue. Trypsin-like enzymes play a physiological role in digestion, coagulation, fibrinolysis, blood pressure regulation, fertility, and inflammation (see: "Design of Enzyme Inhibitors as Drugs" Eds. Sandler, M., Smith, H. J., Oxford Science Publications, 1989). Selective inhibitors of members of this family of enzymes may therefore be useful in the intervention of many disease states. The catalytic mechanism of serine proteinases involves the attack of the active-site serine on the carbonyl bearing the sissile amide bond of the substrate, to give a tetrahedral intermediate. It has been reported that peptide analogs which are stable mimics of this tetrahedral intermediate (i.e., transition-state analogs) can be selective enzyme inhibitors (see Delbaere, L. T. J., Brayer, G. D., J. Mol. Biol. 183:89-103, 1985 and Aoyagi, T., Umezawa, H., Eds., Proteases and Biological Control, Cold Spring Harbor Laboratory Press, 429-454, 1975). Methods for identifying potent and selective inhibitors (as potential drugs) is an active area of research.
Peptide aldehydes were initially discovered as natural products produced by a number of actinomycete strains. Some of these derivatives have bee reported to be selective inhibitors of various types of serine and cysteine proteinases (see Aoyagi, T. et al., cited above). For example, the peptide alaninal elastatinal is a potent elastase inhibitor, while not inhibiting trypsin or trypsin-like enzymes (see Hassall, C. H. et al., FEBS Lett., 183:201-5, 1985). In several cases, the selectivity of these naturally occurring analogs has been enhanced by modifying the sequence. (See, e.g., Bajusz, S. et al., J. Med. Chem. 33:1729-1735, 1990, and McConnell, R. M. et al., J. Med. Chem. 33:86-93, 1990.) Elastase inhibitors are of interest in the treatment of diseases such an emphysema and synthetic peptide aldehydes have been reported to be excellent inhibitors of human leucocyte elastase (see "Design of Enzyme Inhibitors as Drugs" cited above). The peptide arginal leupeptin has been reported to be a selective inhibitor of trypsin-like enzymes (see Aoyagi, T., Umezawa, H., Eds., "Structures and activities of protease inhibitors of microbial origin", Proteases and Biological Control, Cold Spring Harbor Laboratory Press, 429- 454, 1975). Leupeptin, along with naturally occurring variants and synthetic analogs, has been reported to be potent inhibitors of several trypsin like enzymes in the coagulation cascade. Synthetic peptide analogs have been prepared which are reported to show a marked selectivity for particular coagulation factors. For example, one such analog (Me-D-Phe-Pro-Arg-al) has been developed as a thrombin inhibitor and is reported to have significant in vivo anticoagulant activity. (See U.S. Pat. Nos. 4,316,889 (1982), 4,399,065 (1983), 4,478,745 (1984), 4,346,078 (1982), and 4,708,039 (1987).)
Resins for the affinity isolation of specific enzymes have been reported which have unprotected peptide and amino acid aldehydes attached to insoluble supports. (See Patel et al., Biochem, Biophys. Res. Comm. 104, 181-186 (1982) and Patel et al., Biochem. Biophys. Acta, 748, 321-330 (1983).) Those resins were neither intended nor suitable for use in the solid phase synthesis of peptide aldehydes, since the support was attached to the N-terminus of the peptide aldehyde.
Methods for the solution synthesis of peptide aldehydes have been reported. See, e.g., McConnell et al. and references therein; and Bajusz, S. et al. both cited above; Kawamura et al., Chem, Pharm. Bull., 17: 1902 (1969), and Someno et al., Chem. Pharm. Bull. 34, 1748, (1986). The use of semicarbazides as aldehyde protecting reagents for the solution synthesis of peptide aldehydes has also been reported. Westerik and Wolfnden, J. Biol. Chem., 247, 8195 (1972), Ito et al., Chem. Pharm. Bull. 23, 3081, (1975), and McConnell et al. (cited above). The use of a soluble semicarbazide functionalized polymer has been reported for the manual preparation of some peptide aldehydes. Galpin et al., (Pept. Struct. Funct. Proc. Am Pept. Symp., 9th, 799-802 (1985). Edited by: Deber, C. M., Hruby, V. J., Kopple, K. D., Pierce Chem. Co.: Rockford, Ill.). However, such supports were not suitable for the automatic synthesis of peptide aldehydes, since they dissolve in the solvents used for the coupling steps.